WO2024017331A1 - 一种抗肾上腺髓质素非中和抗体、其制备方法及应用 - Google Patents

一种抗肾上腺髓质素非中和抗体、其制备方法及应用 Download PDF

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WO2024017331A1
WO2024017331A1 PCT/CN2023/108389 CN2023108389W WO2024017331A1 WO 2024017331 A1 WO2024017331 A1 WO 2024017331A1 CN 2023108389 W CN2023108389 W CN 2023108389W WO 2024017331 A1 WO2024017331 A1 WO 2024017331A1
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seq
monoclonal antibody
variable region
chain variable
heavy chain
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French (fr)
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郑云程
邓俗俊
杨欣秀
王宗达
顾春银
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上海济煜医药科技有限公司
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
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    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention relates to an anti-adrenomedullin non-neutralizing antibody, its preparation method and application, and belongs to the fields of bioengineering and biotherapy.
  • Adrenomedullin is a 6kDa polypeptide composed of 52 amino acids. It was first discovered in pheochromocytoma in 1993 and was later found to be widely secreted and expressed in vascular epithelial cells and vascular smooth muscle cells. The adrenomedullin gene is located on chromosome 11. After translation, it is gradually enzymatically cleaved by the prohormone to form calcitonin gene-related peptide (CLR). The heterodimer composed of CLR and RAMP2 or RAMP3 is the receptor of adrenomedullin. Based on its receptor-mediated endocytosis and the action of protease, its half-life is only 22 minutes.
  • Adrenomedullin maintains the integrity of endothelial cells and enhances the barrier. This is achieved in the following two ways: (1) enhancing the activity of GTPase Rac1 and increasing the production of cortical actin and stress fibers; (2) reducing myosin light chain kinase-induced actinogenesis by inhibiting the RhoA/ROCK pathway Globulin shrinks. Adrenomedullin also has vasodilation and blood pressure lowering effects.
  • adrenomedullin In healthy people, the concentration of adrenomedullin is extremely low, around 10pg/mL. It can flexibly shuttle inside and outside blood vessels, flexibly regulate vasodilation and maintain endothelial cell barrier function. In patients with septic shock, adrenomedullin concentration is 5-6 times higher than normal, which is directly related to the severity of the disease and prognosis. In patients with septic shock, inflammation-based effects weaken the barrier effect of blood vessels, and vasodilation leads to further reductions in blood pressure.
  • Non-neutralizing antibodies to adrenomedullin restrict adrenomedullin to blood vessels, correct intravascular barrier function, weaken extracellular vasodilation, and increase the half-life of adrenomedullin in plasma to achieve therapeutic and Attenuates the shock symptoms of sepsis. Therefore, there is an urgent need to develop non-neutralizing antibodies with higher affinity for adrenomedullin.
  • one aspect of the present invention provides an anti-adrenomedullin (anti-ADM) monoclonal antibody or a fragment thereof, wherein the monoclonal antibody or fragment specifically binds to the N of human adrenomedullin (ADM).
  • ADM anti-adrenomedullin
  • the N-terminal amino acid sequence 1-21 of the human ADM is as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits a KD value for ADM of less than 10 - 10 M affinity.
  • an “antibody” or “antibody fragment” of the invention is capable of binding ADM, and is therefore directed against ADM, and may therefore be referred to as an "anti-ADM antibody” or “anti-ADM antibody fragment”.
  • the "antibody” or “antibody fragment” of the present invention is a non-neutralizing antibody against adrenomedullin or a fragment thereof.
  • antibodies having "non-neutralizing anti-ADM activity” or antibody fragments that, for example, block less than 80% of the ADM biological activity are collectively referred to as “non-neutralizing” anti-ADM antibodies or antibody fragments.
  • Antibody fragments are defined as:
  • One or more molecules that bind ADM upon addition to a culture of a eukaryotic cell line, reduce the amount of cAMP produced by the cell line by the action of a synthetic human ADM peptide added in parallel, said cell line expressing A functional human recombinant ADM receptor composed of CRLR (calcitonin receptor-like receptor) and RAMP3 (receptor activity modifying protein 3), wherein the added synthetic human ADM is added in such an amount that in the absence of In the case of a non-neutralizing antibody to be analyzed that results in a half-maximal stimulation of cAMP synthesis, binding of said molecule to ADM results in a decrease in cAMP of no more than 80%, even when said non-neutralizing molecule to be analyzed is capable of binding to ADM This is also true when an amount 10 times or more of the amount required to obtain the maximum reduction in cAMP obtained using the non-neutralizing antibody to be analyzed is added.
  • Bioactivity is defined as the effect of a substance that, following its interaction, appears as a living organism or tissue or organ or functional unit in vivo or in vitro (e.g., in an assay). In terms of ADM biological activity, this could be the role of ADM in the human recombinant adrenomedullin receptor cAMP functional assay. Therefore, according to the present invention, biological activity is defined by adrenomedullin receptor cAMP functional assay.
  • Dose response curves were performed with ADM in the human recombinant adrenomedullin receptor cAMP functional assay.
  • the ADM concentration for half-maximal cAMP stimulation can be calculated.
  • ADM concentration dose-response curves (up to a final concentration of 100 ⁇ g/ml) were performed at constant half-maximal cAMP stimulation with ADM-stabilizing antibodies or adrenomedullin-stabilizing antibody fragments, respectively.
  • a 50% maximum inhibition in the ADM bioassay means that the anti-ADM antibody or the anti-adrenomedullin antibody fragment, respectively, blocks 50% of the biological activity of the baseline value.
  • a maximum inhibition of 80% in the ADM bioassay means that the anti-ADM antibody or the anti-adrenomedullin antibody fragment, respectively, blocks 80% of the ADM biological activity. The implication is to block no more than 80% of the biological activity of ADM. This means that approximately 20% of residual ADM biological activity remains.
  • blocking the biological activity of ADM should be understood as only reducing the biological activity of ADM, preferably maximally reducing the ADM biological activity from 100% to 20% of the remaining ADM biological activity, preferably reducing the ADM biological activity.
  • the activity is reduced from 100% to 50% of the remaining ADM biological activity, but in any case there is still ADM biological activity that can be determined as described above.
  • an anti-adrenomedullin (ADM) antibody is an antibody that can specifically bind to ADM
  • an anti-adrenomedullin antibody fragment is a fragment of an ADM antibody, wherein the fragment can specifically bind to ADM.
  • Specific binding to ADM also allows binding of other antigens. This means that the specificity does not preclude the antibody from cross-reacting with polypeptides other than the polypeptide that elicited the antibody. This also applies to the specificity of the anti-ADM antibodies or fragments thereof of the invention.
  • non-neutralizing anti-ADM antibodies or non-neutralizing anti-ADM antibody fragments of the invention provide significant therapeutic advantages over neutralizing anti-ADM antibodies or neutralizing anti-ADM antibody fragments.
  • Antibodies of the present invention are proteins, including one or more polypeptides that are substantially encoded by immunoglobulin genes and can specifically bind to antigens. Recognized immunoglobulin genes include kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes as well as countless immunoglobulins Variable region genes.
  • Full-length immunoglobulin light chain length is typically about 25 KDa or 214 amino acids. The length of a full-length immunoglobulin heavy chain is typically about 50 KDa or 446 amino acids.
  • the light chain is encoded by a variable region gene (about 110 amino acids in length) located at the NH 2 -terminus and a kappa or lambda constant region gene located at the COOH-terminus.
  • the heavy chain is also encoded by a variable region gene (approximately 116 amino acids in length) and one of the other constant region genes.
  • the basic structural unit of an antibody is usually a tetramer consisting of two pairs of identical immunoglobulin chains, each pair having a light chain and a heavy chain. In each pair, the light and heavy chain variable regions bind antigen, while the constant regions mediate effects or functions.
  • Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab and F(ab') 2 as well as bifunctional hybrid and single-chain antibodies.
  • the immunoglobulin light or heavy chain variable region consists of a framework region interrupted by three hypervariable regions, also known as complementarity determining regions (CDR's). As noted above, CDRs are primarily responsible for binding to the epitope of the antigen.
  • Immune complexes are antibodies such as monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibodies or functional antibody fragments that specifically bind to an antigen.
  • CDRs of antibodies can be defined through a variety of methods, such as Kabat definition rules based on sequence variability, Chothia definition rules based on the position of the structural loop region, and concepts based on IMGT ontology (IMGT-ONTOLOGY).
  • the amino acid sequences of VL and VH of the anti-ADM antibody are encoded according to Chothia coding rules, and the light chain CDR1-3 (LCDR 1-3) and heavy chain CDR1-3 (HCDR 1-3) of the anti-ADM antibody are 3) As defined by Chothia.
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed through genetic engineering from immunoglobulin variable and constant region genes belonging to different species.
  • variable segments from mouse monoclonal antibody genes can be linked to human constant segments such as kappa and gamma 1 or gamma 3.
  • a therapeutic chimeric antibody is thus a hybrid protein consisting of a variable domain or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although other mammalian species may be used or by molecular techniques to generate variable regions.
  • a “humanized” immunoglobulin is an immunoglobulin that includes human framework regions and one or more CDRs of a non-human (such as mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin that provides the CDRs is called the "donor” and the human immunoglobulin that provides the framework is called the "acceptor.”
  • all CDRs in the humanized immunoglobulin are from the donor immunoglobulin. Constant regions need not be present, but if present, they must be substantially identical to human immunoglobulin constant regions, that is, at least about 85-90% identical, such as about 95% identical or more. Therefore, all portions of the humanized immunoglobulin, except perhaps the CDRs, are substantially identical to the corresponding portions of the native human immunoglobulin sequence.
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. Humanized antibodies bind the same antigen as the donor antibody that provided the CDRs.
  • the acceptor framework of a humanized immunoglobulin or antibody can have a limited number of substitutions of amino acids from the donor framework. Humanized or other monoclonal antibodies may have other conservative amino acid substitutions that have essentially no effect on antigen binding or other immunoglobulin function.
  • Humanized immunoglobulins can be constructed through genetic engineering.
  • Human antibodies are antibodies in which the light chain and heavy chain genes are of human origin. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortality can be achieved, for example, by EBV infection or by This is accomplished by fusing human B cells with myeloma or hybridoma cells to produce triple hybridoma cells. Human antibodies can also be produced by phage display methods or selected from human combinatorial monoclonal antibody libraries. Human antibodies can also be produced using transgenic animals carrying human immunoglobulin genes
  • anti-ADM antibodies may have forms known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies.
  • the antibody of the invention is a recombinantly produced antibody such as an IgG, or an antibody fragment containing at least the F-variable domain of the heavy and/or light chain such as a chemically coupled antibody (fragment antigen binding) .
  • the anti-adrenomedullin monoclonal antibody fragments of the invention include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di - scFv, VHH and/or dAb.
  • the antibody of the invention can be produced as follows:
  • the synthesized N-terminal 16 amino acid polypeptides of human ADM, the N-terminal 21 amino acid polypeptides of human ADM, or the N-terminal 19 amino acid polypeptides of mouse ADM (referred to as YY-19, SEQ ID NO: 3) and conjugates were used as Immunize mice with the immunogen. Blood was collected one week after the last immunization, and the serum anti-YY-21 titer was determined by ELISA. After the mice with high serum titers were pulse immunized with the immunogen, spleen cells were harvested for fusion. After spleen cell fusion and hybridoma clone screening, hybridoma clones were obtained. Upon affinity determination, the hybridoma clone exhibited an affinity for ADM with a KD value of less than 10 -10 M.
  • KD KD
  • KD KD
  • KD KD
  • kd dissociation rate constant
  • kon association rate constant
  • the heavy chain variable region of the hybridoma clone contained the following CDR sequence:
  • the light chain variable region of the hybridoma clone contains the following CDR sequences:
  • the hybridoma clone comprises the heavy chain variable region sequence shown in SEQ ID NO: 9, and the hybridoma clone comprises the light chain variable region sequence shown in SEQ ID NO: 10 sequence.
  • Humanization of anti-ADM antibodies can be performed according to the following protocol:
  • the human antibody germline genes with the highest homology were selected as the humanization design framework.
  • the heavy chain variable region of the hybridoma clone was subjected to CDR transplantation and back mutation to obtain a humanized heavy chain variable region sequence.
  • the light chain variable region of the hybridoma clone is subjected to CDR transplantation and back mutation to obtain a humanized light chain variable region sequence.
  • the heavy chain variable region of the humanized antibody comprises the following CDR sequences:
  • the light chain variable region of the humanized antibody contains the following CDR sequences:
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 17, and the humanized antibody comprises the heavy chain variable region sequence shown in any of SEQ ID NO: 18 and 19 The light chain variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 17, and the humanized antibody comprises the light chain variable region sequence set forth in SEQ ID NO: 18. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:17, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:19. Variable region sequence.
  • the monoclonal antibody further comprises an antibody light chain constant region, and the light chain constant region is a human kappa chain constant region.
  • the human Kappa chain constant region includes the amino acid sequence shown in SEQ ID NO: 20.
  • the monoclonal antibody further comprises an antibody heavy chain constant region, which is a human IgGl constant region.
  • the human IgG1 constant region includes the amino acid sequence shown in SEQ ID NO: 21.
  • the light chain variable region sequence of the monoclonal antibody of the present invention is combined with the human Kappa chain constant region to form an antibody light chain
  • the heavy chain variable region sequence of the monoclonal antibody of the present invention is combined with the human IgG1 constant region to form an anti- Weight chain.
  • the monoclonal antibody comprises the heavy chain sequence shown in any one of SEQ ID NO:23, 25, and 27, and the monoclonal antibody comprises SEQ ID NO:22, The light chain sequence shown in any one of 24 and 26.
  • the monoclonal antibody comprises the heavy chain sequence shown in SEQ ID NO:23, and the monoclonal antibody comprises the light chain sequence shown in SEQ ID NO:22.
  • the monoclonal antibody comprises the heavy chain sequence shown in SEQ ID NO:25, and the monoclonal antibody comprises the light chain sequence shown in SEQ ID NO:24.
  • the monoclonal antibody comprises the heavy chain sequence set forth in SEQ ID NO:27, and the monoclonal antibody comprises the light chain sequence set forth in SEQ ID NO:26.
  • the double plasmids are co-transfected into Expi293F or CHO-K1 cells for transient expression of the antibody.
  • the antibody is purified through a Protein A affinity chromatography column. Upon affinity determination, the humanized anti-ADM antibody exhibited an affinity for ADM with a KD value of less than 10 -10 M.
  • the present invention can also perform anti-ADM screening by selecting a fully human single-chain phage antibody library. Screening of monoclonal antibodies.
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • “monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies making up the population are identical except for possible naturally occurring mutations and/or translations that may be present in minimal amounts.
  • post-modification such as isomerization, amidation
  • the anti-adrenomedullin monoclonal antibody contains a post-translational modification (PTM) site for NG.
  • PTM post-translational modification
  • the post-translational modification (PTM) site of NG contained in the anti-adrenomedullin monoclonal antibody is site-directed mutated into QG to eliminate deamidation isomerization.
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the monoclonal antibody comprises the heavy chain variable region sequence shown in either SEQ ID NO: 28 and 29, and the monoclonal antibody comprises the light chain variable region sequence shown in SEQ ID NO: 35. Chain variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 28, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 35. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:29, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:35. Variable region sequence.
  • the present invention In order to improve the affinity of fully human antibodies with human ADM, the present invention also designed and constructed an affinity mature library of fully human antibodies. Through the screening of fully human antibody affinity mature libraries and the identification of monoclones, the present invention obtained different fully human anti-ADM antibodies, all of which exhibit an affinity for ADM with a KD value of less than 10 -10 M.
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the anti-adrenomedullin monoclonal antibody or a fragment thereof is provided.
  • the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the light chain variable region of the monoclonal antibody includes the following CDR sequence:
  • the present invention also obtains the heavy chain variable region sequence and light chain variable region sequence of the antibody with improved affinity.
  • the monoclonal antibody comprises the heavy chain shown in any one of SEQ ID NO: 30 to 34 Variable region sequence
  • the monoclonal antibody includes the light chain variable region sequence shown in any one of SEQ ID NO: 35 to 42.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:30, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:35. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 36. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence set forth in SEQ ID NO: 37. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 32, and the humanized antibody comprises the light chain variable region sequence set forth in SEQ ID NO: 36. Changing area order List.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:33, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:38. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 38. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 39. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:34, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:38. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 34, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 40. Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 33, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 41 Variable region sequence.
  • the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 42. Variable region sequence.
  • the monoclonal antibody further comprises an antibody light chain constant region, and the light chain constant region is a human kappa chain constant region.
  • the human Kappa chain constant region includes the amino acid sequence shown in SEQ ID NO: 20.
  • the monoclonal antibody further comprises an antibody heavy chain constant region,
  • the heavy chain constant region is a human IgG1 constant region.
  • the human IgG1 constant region includes the amino acid sequence shown in SEQ ID NO: 21.
  • the light chain variable region sequence of the monoclonal antibody is combined with the human Kappa chain constant region to form an antibody light chain
  • the heavy chain variable region sequence of the monoclonal antibody is combined with the human IgG1 constant region to form an antibody heavy chain .
  • the synthesized gene fragment is cloned into the expression vector pcDNA3.4, expressed and purified in CHO cells, and affinity matured monoclonal antibodies can be obtained.
  • the anti-ADM antibody exhibits an affinity for ADM with a KD value of less than 10 -10 M as determined by affinity determination.
  • the present invention provides a nucleotide sequence encoding the anti-adrenomedullin monoclonal antibody of the present invention or a fragment thereof.
  • Another aspect of the present invention provides an expression vector comprising the nucleotide sequence of the present invention, preferably the vector pcDNA3.4.
  • Another aspect of the present invention provides a host cell comprising the nucleotide sequence or expression vector of the present invention, which is preferably a CHO cell.
  • Another aspect of the present invention provides a pharmaceutical composition, detection reagent or kit containing the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention.
  • Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of drugs for maintaining endothelial cell integrity and enhancing barrier function.
  • Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of drugs for controlling vasodilation and lowering blood pressure.
  • Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of medicaments for the treatment or prevention of septic shock or traumatic injury, especially in the late stage of sepsis.
  • Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of a medicament for reducing the risk of death of a patient with a chronic or acute disease or an acute disease, the chronic or acute disease being or acute illness selected from severe infections such as meningitis, systemic inflammatory response Syndrome (SIRS), sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, edema; shock such as septic shock and organ dysfunction such as renal dysfunction, Liver dysfunction, burns, surgery, trauma, poisoning.
  • severe infections such as meningitis, systemic inflammatory response Syndrome (SIRS), sepsis
  • other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, edema
  • shock such as septic shock and organ dysfunction such as renal dysfunction, Liver dysfunction,
  • Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of medicaments for stabilizing the circulation, especially the systemic circulation, of patients suffering from chronic or acute diseases or acute Conditions
  • the chronic or acute diseases or acute conditions are selected from severe infections such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis; other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke , atherosclerosis, edema; shock such as septic shock and organ dysfunction such as renal dysfunction, liver dysfunction, burns, surgery, trauma, and poisoning.
  • the anti-adrenomedullin monoclonal antibody or fragment thereof is used in combination with another agent.
  • the pharmaceutical agent is selected from the group consisting of vasopressors, intravenous solutions, TNF- ⁇ -antibodies and antibiotics.
  • the present invention screens out anti-ADM monoclonal antibodies through hybridoma technology, and obtains fully human anti-ADM monoclonal antibodies through humanized expression of antibodies and panning of a fully human single-chain phage antibody library. On this basis, the present invention screened the affinity maturation library and finally obtained a drug with higher activity of binding to the N-terminus of human ADM. Compared with anti-ADM non-neutralizing antibodies existing in the prior art, the anti-ADM non-neutralizing antibodies of the present invention exhibit an affinity for ADM with a KD value of less than 10 -10 M.
  • Figure 1 shows that anti-ADM antibody blocks the production of CHOK1/CRLR/RAMP3 cells induced by hADM.
  • Figure 2 is a graph showing the results of anti-ADM antibody survival rate in LPS-induced sepsis in mice.
  • Example 1 Screening of anti-ADM monoclones using hybridoma technology
  • the synthesized N-terminal 16 amino acid polypeptide of human ADM (referred to as YY-16, SEQ ID NO: 1), the N-terminal 21 amino acid polypeptide of human ADM (referred to as YY-21, SEQ ID NO: 2) or mouse ADM
  • the conjugate of the N-terminal 19 amino acid peptide (referred to as YY-19, SEQ ID NO: 3) and KLH (Sigma, H8283) was used as an immunogen to immunize mice.
  • the immunogen and Freund's complete adjuvant were emulsified at a ratio of 1:1, and intraperitoneally injected into 6 to 8-week-old female Balb/c mice, SJL mice or SD rats at 100 ⁇ g/mouse; thereafter Booster immunizations were performed every 2 to 3 weeks with 50 ⁇ g of immunogen per animal plus Freund's incomplete adjuvant.
  • blood was collected to measure the titer. Blood was collected one week after the last immunization, and the serum anti-YY-21 titer was determined by ELISA. After the mice with high serum titer were pulse immunized by intraperitoneal injection of 50 ⁇ g of immunogen, the spleen cells of the animals were harvested for fusion on the third day.
  • mice After the mice were euthanized, the cells were dissected, the spleens were removed, the cells were ground and collected, and the cells were suspended in 5 mL of red blood cell lysis buffer and placed at 4°C for 5 min. The reaction was terminated with DMEM+10% FBS. After centrifugation, resuspend the splenocytes in 40 mL DMEM, let stand for 2 to 3 minutes, and then transfer the supernatant to another 50 mL centrifuge tube.
  • hybridoma clone 40E12 was screened, the cell line was cryopreserved, and 50 mL small-scale production was carried out using serum-free medium. The protein A column was purified for subsequent identification.
  • Octet RED96e was used to determine candidate antibodies and biotin-labeled human ADM (referred to as human ADM-C-biotin, the human ADM sequence is shown in SEQ ID NO: 4) and mouse ADM (referred to as mouse ADM-C-biotin, mouse The affinity of the ADM sequence (shown in SEQ ID NO:5). Both the antigen and antibody were diluted with 1 ⁇ PBST, the antigen concentration was 2 ⁇ g/ml, and the antibody working concentration was 100 nM.
  • the sample was added to a 96-well plate (Greiner bio-one, 655209), and the system was 200 ⁇ L/well. Then set the software parameters, the plate temperature is set to 30°C, and the frequency of collecting standard kinetic signals is 5.0HZ.
  • prewet the streptavidin sensor (Fortébio, Cat. No.: 18-5020) with 1 ⁇ PBST for 10 minutes, and then put it on the machine for detection.
  • Each cycle includes the following steps: 1) Immerse in the buffer for 180 seconds to stabilize the baseline; 2) Solidify the antigen for 10 seconds to bind the antigen to the sensor, and control the antigen binding amount between 0.5-1.0nm; 3) Immerse the sensor into the buffer for 180 seconds ;4) Antigen and antibody combine. When combined, 180s; 5) Dissociation of antigen and antibody, time 10min.
  • VH primers sequences such as SEQ ID NO: 6
  • VK primer sequence shown in SEQ ID NO: 7
  • polyC primer sequence shown in SEQ ID NO: 8
  • the light and heavy chain variable regions were Chothia numbered.
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 of clone 40E12 defined by Chothia are shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 respectively.
  • LCDR1, LCDR2 and The amino acid sequences of LCDR3 are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively.
  • human antibody germline genes with the highest homology were selected as the humanization design framework.
  • the heavy chain variable region is based on the human antibody germline gene sequences IGHV7-4-1*02 and IGHJ6*01 as the framework, and 40E12VH (also referred to as 2004hzVH0) is subjected to CDR transplantation and reverse mutation to obtain the humanized heavy chain variable region sequence.
  • 2004hzVH9 amino acid sequence such as SEQ ID NO: 17).
  • the light chain variable region uses the human antibody germline gene sequences IGKV2-30*02 and IGKJ2*01 as the framework, and combines 40E12VK (also referred to as 2004hzVK0) humanized light chain variable region sequences 2004hzVK7 (amino acid sequence such as SEQ ID NO: 18) and 2004hzVK9 (amino acid sequence such as SEQ ID NO: 19) subjected to CDR transplantation and backmutation.
  • 40E12VK also referred to as 2004hzVK0
  • 2004hzVK7 amino acid sequence
  • 2004hzVK9 amino acid sequence such as SEQ ID NO: 19
  • the 2004hzVK7 and 2004hzVK9 sequences were combined with the human Kappa chain constant region (CL, amino acid sequence SEQ ID NO: 20) to form an antibody light chain, and the 2004hzVH9 sequence was combined with the human IgG1 constant region (CH, amino acid sequence SEQ ID NO: 21) to form an antibody Weight chain.
  • 2004hzVH0 and 2004hzVK0 were combined with the human heavy chain constant region and the human light chain constant region respectively and then paired to form the chimeric antibody 2004hz00 (sequences are shown in SEQ ID NO: 22 and 23); 2004hzVH9 and 2004hzVK7 were combined with the human heavy chain constant region and the human light chain constant region respectively.
  • the light chain constant regions were combined and paired to form humanized antibody 2004hz97 (sequences are shown in SEQ ID NO: 24 and 25); 2004hzVH9 and 2004hzVK9 were combined with human heavy chain constant regions and human light chain constant regions respectively to form humanized antibodies.
  • Antibody 2004hz99 sequences are shown in SEQ ID NO: 26 and 27).
  • Octet RED96e (Fortebio) was used to determine the affinity of antibodies 2004hz00, 2004hz97, 2004hz99 and biotin-labeled human and mouse ADM (human ADM product number: 894757, mouse ADM product number: 894758, synthesized by GLBiochem). Both antigens and antibodies were used 1 ⁇ PBST (1 ⁇ PBS: Sangon, B548117-0500; 0.02% Tween 20: sigma, P1379) was diluted, the antibody concentration was 100 nM, and the antigen concentration was 2 ⁇ g/mL.
  • Each cycle includes the following steps: 1) Immerse in the buffer for 60 seconds; 2) Detect whether the antigen binds non-specifically to the sensor; 3) Regenerate the 10mM glycine solution at pH 1.7; 4) Immerse in the buffer for 60 seconds; 5) The antigen is solidified on the sensor on, the time is 10s; 6) the sensor is immersed in the buffer for 180s; 7) the antigen and antibody are combined, the time is 180s; 8) the antigen-antibody dissociation, the time is 600s; 9) the sensor is regenerated.
  • humanized antibodies 2004hz97 and 2004hz99 have good physical and chemical properties after one-step protein A purification.
  • Example 5 Panning of fully human single-chain phage antibody library
  • the antigen-specifically bound phages were eluted with 450 ⁇ L of 100 mM hydrochloric acid, and 50 ⁇ L of 1M Tris-HCl at pH 11 was added to neutralize and infect E. coli SS320 in the logarithmic growth phase, and the phages were generated and purified for the next round of screening.
  • the screening method was the same as in the first round, except that the amount of antigen was reduced to 4 ⁇ g.
  • the enriched phages after two rounds of screening were used to identify the enrichment status using enzyme-linked immunoassay (ELISA). The results showed that the phages were significantly enriched after two rounds of panning.
  • adrenomedullin bioassay The effect of selected anti-ADM antibodies on ADM biological activity was tested in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay).
  • the anti-ADM antibody was diluted to 1600 ⁇ g/mL with Stimulation Buffer 1 (Cisbio, 64SB1FDD), with a working concentration of 400 ⁇ g/mL, followed by 3-fold gradient dilution with Stimulation Buffer 1 (8 ⁇ L + 16 ⁇ L Stimulation Buffer 1), in which the antagonist human ADM (22-52) (Alfa Aesar, Cat. No.: 159899-65-7) was diluted to 12000 ⁇ g/mL, with a working concentration of 3000 ⁇ g/mL, and then 2.5 ⁇ L of anti-ADM antibody was added to the corresponding well in the experimental plate. Dilute human ADM JMB2004 YY-52 protein (Gill Biochemical, Cat.
  • CHO-K1/CRLR/RAMP3 human recombinant adrenomedullin receptor
  • the cAMP content was detected using the HTRF kit (Cisbio, 62AM4PEB). That is, 5 ⁇ L of cAMP-d2 reagent working solution was added to the experimental well, and then 5 ⁇ L of cAMP Eu-Cryptate antibody working solution was added. After incubation at room temperature for 1 hour, the HTRF value was detected by a microplate reader.
  • the fully human antibody 1F12 obtained through screening was affinity matured to improve its affinity with human ADM.
  • the heavy chain antigen-binding determinant 2 (CDR_H2) of 1F12 contains the post-translational modification (PTM) site of NG, site-directed mutation into QG eliminates deamidation isomerization, and the mutated antibody is named 1F12PTM ⁇ (variable region amino acid Sequences are shown in SEQ ID NO: 29 and 35).
  • coding was performed according to Chothia rules, the CDR region was defined according to Chothia, and the amino acids at the HCDR-3 and LCDR-3 sites were randomly mutated to construct a mutation library.
  • NNK mutation primers were designed to perform polymerase chain reaction (PCR) to amplify the HCDR-3 and LCDR-3 mutation library gene fragments.
  • PCR polymerase chain reaction
  • the amplified VH and VL gene fragments were recovered and electroporated together with the yeast display plasmid into the Saccharomyces cerevisiae strain EBY100 ( Purchased from ATCC), the VH and VL genes were inserted into the yeast display plasmid through homologous recombination of Saccharomyces cerevisiae, thereby realizing a Fab mutation library that displays antibodies on the yeast cell wall surface.
  • the library was named JYYDL196-197.
  • the library JYYDL196-197 was cultured in 250 mL of SD-Trp-Leu liquid medium (Clontech, Cat. No.: 630316) at 30°C overnight; 1.0 ⁇ 10 9 bacteria were taken from each and resuspended in 200 mL of YPGP induction medium (2% Galactose, 2% peptone, 1% yeast extract, 0.54% Na 2 HPO 4 , 0.86% NaH 2 PO 4 ⁇ H 2 O), cultured at 20°C for 24h, and placed at 4°C until use.
  • the sequence of the parent 1F12PTM ⁇ was displayed on the yeast surface and used as a parental control.
  • Ab2004.Am01 continues to undergo affinity maturation to improve its affinity with human and mouse ADM.
  • the amino acids of HCDR-1, HCDR-2, LCDRL-1, LCDR-2, and LCDR-3 of Ab2004.Am01 were selected for mutation library construction, and the library number was JYYDL208-212.
  • the library JYYDL208-212 was cultured and induced before use.
  • the Fab sequence of Ab2004.Am01 was displayed on the yeast surface and used as a parental control for this round of affinity maturation.
  • JYYDL208-209 the second and third round screening products of JYYDL211, selected several single clones from each to construct the light and heavy chain mutation combinatorial library JYYDL227.
  • .Am32 (the amino acid sequence of the variable region is shown in SEQ ID NO: 31 and 37), Ab2004.Am33 (the amino acid sequence of the variable region is shown in SEQ ID NO: 32 and 36), Ab2004.Am34 (the amino acid sequence of the variable region is shown in SEQ ID NO: 32 and 36) As shown in SEQ ID NO:33 and 38), Ab2004.Am35 (the amino acid sequence of the variable region is as shown in SEQ ID NO:31 and 38), Ab2004.Am36 (the amino acid sequence of the variable region is as shown in SEQ ID NO:31 and 39) shown), Ab2004.Am37 (the variable region amino acid sequence is shown in SEQ ID NO: 34 and 38), Ab2004.Am38 (the amino acid sequence of the variable region is shown in SEQ ID NO:34 and 40), Ab2004.Am39 (the amino acid sequence of the variable region is shown in SEQ ID NO:33 and 41) and Ab2004.Am40 (the amino acid sequence of the variable region is shown in SEQ ID NO:33 and 41) The sequences are shown in
  • Example 2.2 the VH and VK sequences of each clone were combined with the human Kappa chain constant region (CL, amino acid sequence SEQ ID NO: 20) and the human IgG1 constant region (CH, amino acid sequence SEQ ID NO: 21) respectively to form an antibody.
  • the light chain and heavy chain were loaded into the expression vector pcDNA3.4 (Life Technologies), entrusted Nanjing GenScript Biotechnology Co., Ltd. to perform transient CHO cell expression and purification, and finally obtained the affinity mature candidate antibodies as shown in Table 9.
  • Example 3 continue to measure the affinity of the affinity mature antibodies Ab2004.Am31-Ab2004.Am40 and the parent antibody Ab2004.Am01 to human and mouse ADM respectively.
  • the results are shown in Table 10.
  • Table 10 the affinity of Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 to human ADM is the most improved compared to the parent antibody Ab2004.Am01, and the affinity to mouse ADM is comparable to the positive control antibody Enibarcimab.
  • Example 14 Evaluation of physicochemical properties of candidate antibodies
  • Example 15 Pharmacodynamic study of ADM antibody in LPS-induced C57BL/6J mouse sepsis model
  • C57BL/6J mice were purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd., 10-11 weeks old, male, 50 in total.

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Abstract

本发明属于生物工程和生物治疗领域，公开了一种抗ADM单克隆抗体或其片段，以及包含所述单克隆抗体或其片段的药物组合物、检测试剂或套装药盒，本发明还公开了所述单克隆抗体或其片段在制备药物中的应用。

Description

一种抗肾上腺髓质素非中和抗体、其制备方法及应用
本申请要求享有于2022年7月20日向中国国家知识产权局提交的，专利申请号为202210863441.9，名称为“一种抗肾上腺髓质素非中和抗体、其制备方法及应用”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及一种抗肾上腺髓质素非中和抗体、其制备方法及应用，属于生物工程和生物治疗领域。
背景技术
肾上腺髓质素(Adrenomedullin，ADM)是一段由52个氨基酸组成的6kDa的多肽，1993年最早在嗜铬细胞瘤中发现，后发现在血管表皮细胞及血管平滑肌细胞均广泛分泌表达。肾上腺髓质素的基因位于11号染色体上，翻译后由前激素原逐步酶切，形成作为降钙素基因相关肽(CLR)。CLR与RAMP2或RAMP3组成的异源二聚体是肾上腺髓质素的受体，基于其受体介导的内吞作用以及蛋白酶的作用，其半衰期仅为22min。
肾上腺髓质素具有维持内皮细胞完整性，增强屏障的作用。这是通过以下两方面实现的：(1)加强GTPase Rac1的活性，提高皮层肌动蛋白和应力纤维的生成；(2)通过抑制RhoA/ROCK途径，减少肌球蛋白轻链激酶诱导的肌动球蛋白收缩。肾上腺髓质素还具有血管舒张，降低血压的作用。这是通过以下两方面实现的：(1)通过PI3K/Akt途径，释放NO，活化环磷酸鸟苷(cGMP)/激活蛋白激酶K(PKG)；(2)通过与血管平滑肌的结合，导致环 磷酸腺苷(cAMP)/激活蛋白激酶A(PKA)浓度提高，通过磷酸化作用，引起平滑肌细胞的松弛。
在健康人群中，肾上腺髓质素浓度极低，在10pg/mL左右，可以灵活穿梭于血管内外，灵活调节血管舒张和维持内皮细胞屏障功能。在脓毒症休克患者中，肾上腺髓质素浓度是正常情况下的5-6倍，和病情严重程度和预后直接相关。在脓毒症休克患者中，基于炎症的作用减弱了血管的屏障作用，血管舒张导致血压进一步降低。
肾上腺髓质素非中和抗体将肾上腺髓质素限制于血管内，纠正了血管内屏障功能，减弱了胞外的血管舒张作用，提高了肾上腺髓质素在血浆内的半衰期，以达到治疗及减弱脓毒症的休克症状。因此，目前亟需研制能与肾上腺髓质素具有较高亲和力的非中和抗体。
发明内容
针对上述技术问题，本发明一方面提供了一种抗肾上腺髓质素(抗ADM)单克隆抗体或其片段，其中所述单克隆抗体或片段特异性结合人肾上腺髓质素(ADM)的N端第1-21个氨基酸序列，所述人ADM的N端第1-21位氨基酸序列如SEQ ID NO：2所示，并且所述单克隆抗体或片段展现出对ADM的KD值小于10-10M的亲和力。
在本发明的整篇说明书中，本发明的“抗体”或“抗体片段”能够结合ADM，因此针对ADM，并因此可被称为“抗ADM抗体”或“抗ADM抗体片段”。
本发明所述“抗体”或“抗体片段”是抗肾上腺髓质素的非中和抗体或其片段。
在这种背景下，出于简化的目的，统称为“非中和”抗ADM抗体或抗体片段(其例如封闭80％以下的ADM生物活性)的具有“非中和抗ADM活性”的抗体或抗体片段被定义为：
结合ADM的一种或多种分子，在添加至真核细胞系培养物之后，通过平行添加的合成的人ADM肽的作用降低所述细胞系所产生的cAMP的量，所述细胞系表达由CRLR(降钙素受体样受体)和RAMP3(受体活性修饰蛋白3)组成的功能性人重组ADM受体，其中所述添加的合成的人ADM以这样的量添加：在不存在待分析的非中和抗体的情况下，导致cAMP合成的半最大刺激，其中所述分子结合ADM导致cAMP降低的程度不超过80％，甚至当待分析的能与ADM结合的所述非中和分子以所述量(用待分析的非中和抗体能得到的获得cAMP的最大降低所需的)10倍以上的量添加时也如此。
相同的定义适用于其他范围：95％、90％、50％等。
生物活性被定义为这样的作用：一种物质在其相互作用后在体内或体外(例如，在测定中)呈现为活的生物体或组织或器官或功能单元。就ADM生物活性而言，这可以是ADM在人重组肾上腺髓质素受体cAMP功能测定中的作用。因此，根据本发明，生物活性通过肾上腺髓质素受体cAMP功能测定来定义。
为了在这样的测定中测定ADM生物活性，可以进行以下步骤：
在所述人重组肾上腺髓质素受体cAMP功能测定中用ADM进行剂量反应曲线。
可以计算半最大cAMP刺激的ADM浓度。
在恒定的半最大cAMP刺激时，分别通过ADM稳定抗体或肾上腺髓质素稳定抗体片段进行ADM浓度剂量反应曲线(高达100μg/ml的终浓度)。
所述ADM生物测定中50％的最大抑制表示，所述抗ADM抗体或所述抗肾上腺髓质素抗体片段分别封闭基线值50％的生物活性。所述ADM生物测定中80％的最大抑制表示，所述抗ADM抗体或所述抗肾上腺髓质素抗体片段分别封闭80％的ADM生物活性。其含义是封闭不超过80％的ADM生物活性。这意味着仍存在约20％的残余ADM生物活性。
然而，通过本说明书以及在上述背景下，结合本文公开的抗ADM抗体和 抗ADM抗体片段而言，表述“封闭ADM的生物活性”应当被理解为仅降低ADM的生物活性，优选最大时将ADM生物活性从100％降低至20％的剩余ADM生物活性，优选将ADM生物活性从100％降低至50％的剩余ADM生物活性，但是在任何情况下，仍有可以如上所述测定的ADM生物活性。
本文中，抗肾上腺髓质素(ADM)抗体是能特异性结合ADM的抗体，抗肾上腺髓质素抗体片段是ADM抗体的片段，其中所述片段能特异性结合ADM。特异性结合ADM也允许结合其他抗原。这意味着，该特异性不排除该抗体可以与激发该抗体的多肽之外的多肽交叉反应。这也适合本发明的抗ADM抗体或其片段的特异性。
本发明的非中和抗ADM抗体或非中和抗ADM抗体片段提供显著超越中和抗ADM抗体或中和抗ADM抗体片段的治疗优势。
本发明的抗体是蛋白，包括实质上由免疫球蛋白基因编码的能特异性结合抗原的一种或多种多肽。公认的免疫球蛋白基因包括κ、λ、α(IgA)、γ(IgG1，IgG2，IgG3，IgG4)、δ(IgD)、ε(IgE)和μ(IgM)恒定区基因以及无数的免疫球蛋白可变区基因。全长免疫球蛋白轻链长度通常为约25KDa或214个氨基酸。全长免疫球蛋白重链的长度通常为约50KDa或446个氨基酸。轻链由位于NH2-端的可变区基因(长度约110个氨基酸)和位于COOH-端的κ或λ恒定区基因编码。重链同样由可变区基因(长度约116个氨基酸)和其他恒定区基因之一编码。
抗体的基本结构单元通常是由两对相同的免疫球蛋白链组成的四聚体，每一对都具有一条轻链和一条重链。在每对中，轻链和重链可变区结合抗原，而恒定区介导效应或功能。免疫球蛋白还以多种其他形式存在，包括例如，Fv、Fab和F(ab')2以及双功能杂合抗体和单链抗体。免疫球蛋白轻链或重链可变区包括由三个高变区中断的框架区，所述高变区也称为互补决定区(CDR's)。如上文所指出的，CDR主要负责结合抗原的表位。免疫复合物是抗体如单克隆抗体、嵌合抗体人源化抗体或人抗体或功能性抗体片段特异性结合抗原。
在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则、基于结构环区域位置的Chothia定义规则和基于IMGT本体论(IMGT-ONTOLOGY)的概念。本发明中，所述抗ADM抗体的VL和VH的氨基酸序列按Chothia编码规则进行编码，所述抗ADM抗体的轻链CDR1-3(LCDR 1-3)和重链CDR1-3(HCDR 1-3)按Chothia定义。
嵌合抗体是这样的抗体：通过基因工程从属于不同物种的免疫球蛋白可变区和恒定区基因构建其轻链和重链基因。例如，可以将来自小鼠单克隆抗体基因的可变节段连接至人恒定节段如κ和γ1或γ3。在一个实例中，治疗性嵌合抗体因而是由来自小鼠抗体的可变结构域或抗原结合结构域和来自人抗体的恒定或效应结构域组成的杂合蛋白，尽管可以使用其他哺乳动物物种或通过分子技术产生可变区。
“人源化”免疫球蛋白是包括人框架区和非人(诸如小鼠、大鼠或合成的)免疫球蛋白的一个或多个CDR的免疫球蛋白。提供CDR的非人免疫球蛋白称为“供体”，提供框架的人免疫球蛋白称为“受体”。
在一个实施方案中，人源化免疫球蛋白中的所有CDR都来自供体免疫球蛋白。不需要存在恒定区，但是如果存在，它们必须与人免疫球蛋白恒定区基本一致，即至少约85-90％，诸如约95％一致或更加一致。因此，人源化免疫球蛋白的所有部分，可能除了CDR，都与天然人免疫球蛋白序列的对应部分基本一致。“人源化抗体”是包含人源化轻链和人源化重链免疫球蛋白的抗体。人源化抗体能与提供CDR的供体抗体结合相同的抗原。人源化免疫球蛋白或抗体的受体框架可以具有有限数量的来自供体框架的氨基酸的取代。人源化或其他单克隆抗体可以具有其他保守的氨基酸取代，其对抗原结合或其他免疫球蛋白功能基本上没有影响。人源化免疫球蛋白可以通过基因工程的手段来构建。
人抗体是这样的抗体：其中轻链和重链基因来自人。人抗体可以通过使分泌目的抗体的人B细胞永生化来产生。永生化可以例如通过EBV感染或通过 将人B细胞与骨髓瘤或杂交瘤细胞融合以产生三源杂交瘤细胞来实现。人抗体还可以通过噬菌体展示方法产生，或从人组合单克隆抗体文库选择。人抗体还可以利用携带人免疫球蛋白基因的转基因动物来制备
因此，抗ADM抗体可以具有本领域内已知的形式。实例为人抗体、单克隆抗体、人源化抗体、嵌合抗体、CDR移植抗体。
在优选的实施方案中，本发明的抗体是重组产生的抗体例如IgG，或至少含有重链和/或轻链的F-可变结构域的抗体片段例如化学偶联的抗体(片段抗原结合)。
因此，在本发明优选的实施方案中，本发明所述的抗肾上腺髓质素单克隆抗体片段包括Fab，Fab'，F(ab)2、Fv片段、F(ab')2、scFv、di-scFv、VHH和/或dAb。
在本发明一个优选的实施方案中，本发明的抗体可以如下产生：
分别将合成的人ADM的N端16个氨基酸多肽、人ADM的N端21个氨基酸多肽或鼠ADM的N端19个氨基酸多肽(简称YY-19，SEQ ID NO:3)与偶联物作为免疫原免疫小鼠。最后一次免疫一周后取血，ELISA测定血清抗YY-21的滴度。血清效价高的鼠经免疫原冲击免疫后，取脾脏细胞进行融合。经脾细胞融合和杂交瘤克隆筛选后，得到杂交瘤克隆。经亲和力测定，杂交瘤克隆展现出对ADM的KD值小于10-10M的亲和力。
本发明中，术语“KD”、“KD”或“KD”可互换使用，通常是指抗体-抗原相互作用的平衡解离常数。本发明中使用的“KD”是解离速率常数(kdis，也称为“解离率(off-rate)(koff)”或“kd”)与结合速率常数(kon，也称为“结合率(kon)”或“ka”)的比值。
经测序，所述杂交瘤克隆的重链可变区包含以下CDR序列：
(i)GYAFTTF(如SEQ ID NO:11所示)，
(ii)NTYSRV(如SEQ ID NO:12所示)，
(iii)GYGGEGGLGF(如SEQ ID NO:13所示)，以及
所述杂交瘤克隆的轻链可变区包含以下CDR序列：
(i)RSSQSIIDSDGNTYLE(如SEQ ID NO:14所示)，
(ii)KVSNRFS(如SEQ ID NO:15所示)，
(iii)FQGSHFPYT(如SEQ ID NO:16所示)。
在本发明的某些实施方案中，所述杂交瘤克隆包含SEQ ID NO:9所示的重链可变区序列，所述杂交瘤克隆包含SEQ ID NO:10所示的轻链可变区序列。
可以按照以下方案进行抗ADM抗体的人源化：
通过序列比对，挑选同源性最高的人抗体胚系基因作为人源化设计框架。将杂交瘤克隆的重链可变区进行CDR移植和回复突变获得人源化重链可变区序列。将杂交瘤克隆的轻链可变区进行CDR移植和回复突变获得人源化轻链可变区序列。
由此，在本发明的某些实施方案中，所述人源化抗体的重链可变区包含以下CDR序列：
(i)GYAFTTF(如SEQ ID NO:11所示)，
(ii)NTYSRV(如SEQ ID NO:12所示)，
(iii)GYGGEGGLGF(如SEQ ID NO:13所示)，以及
所述人源化抗体的轻链可变区包含以下CDR序列：
(i)RSSQSIIDSDGNTYLE(如SEQ ID NO:14所示)，
(ii)KVSNRFS(如SEQ ID NO:15所示)，
(iii)FQGSHFPYT(如SEQ ID NO:16所示)。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:17所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:18和19任一所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:17所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:18所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:17所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:19所示的轻链可变区序列。
在本发明的某些实施方案中，所述单克隆抗体还包含抗体轻链恒定区，所述轻链恒定区为人Kappa链恒定区。在本发明中，所述人Kappa链恒定区包含SEQ ID NO：20所示的氨基酸序列。
在本发明的某些实施方案中，所述单克隆抗体还包含抗体重链恒定区，所述重链恒定区为人IgG1恒定区。在本发明中，所述人IgG1恒定区包含SEQ ID NO：21所示的氨基酸序列。
将本发明所述单克隆抗体的轻链可变区序列与人Kappa链恒定区组合成抗体轻链，将本发明所述单克隆抗体的重链可变区序列与人IgG1恒定区组合成抗体重链。
由此，在本发明的某些实施方案中，所述单克隆抗体包含SEQ ID NO:23、25、27任一所示的重链序列，所述单克隆抗体抗体包含SEQ ID NO:22、24、26任一所示的轻链序列。
在本发明的某些实施方案中，所述单克隆抗体包含SEQ ID NO:23所示的重链序列，所述单克隆抗体包含SEQ ID NO:22所示的轻链序列。
在本发明的某些实施方案中，所述单克隆抗体包含SEQ ID NO:25所示的重链序列，所述单克隆抗体包含SEQ ID NO:24所示的轻链序列。
在本发明的某些实施方案中，所述单克隆抗体包含SEQ ID NO:27所示的重链序列，所述单克隆抗体包含SEQ ID NO:26所示的轻链序列。
密码子优化后进行基因合成，克隆合成后基因片段到表达载体中。表达质粒扩增和质粒抽提后双质粒共转Expi293F或CHO-K1细胞中，进行抗体瞬转表达，表达后通过Protein A亲和层析柱纯化。经亲和力测定，人源化抗ADM抗体展现出对ADM的KD值小于10-10M的亲和力。
本发明还可以通过对全人源单链噬菌体抗体库进行陶选，来进行抗ADM 单克隆抗体的筛选。
由此，在本发明优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTSY(如SEQ ID NO:43所示)，
(ii)SAYNGN(如SEQ ID NO:44所示)，
(iii)EGRSGGSFDI(如SEQ ID NO:45所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DASNLET(如SEQ ID NO:47所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本申请中，“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即构成群体的各个抗体相同，除了可能以极小量存在的可能的天然存在突变和/或翻译后修饰(例如异构化、酰胺化)外。例如，在本发明的某些实施方案中，所述的抗肾上腺髓质素单克隆抗体含有NG的翻译后修饰(PTM)位点。
在本发明的某些实施方案中，将所述的抗肾上腺髓质素单克隆抗体含有的NG的翻译后修饰(PTM)位点，定点突变成QG去除脱酰胺异构作用。
由此，在本发明的某些实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTSY(如SEQ ID NO:43所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRSGGSFDI(如SEQ ID NO:45所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DASNLET(如SEQ ID NO:47所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明的某些实施方案中，所述单克隆抗体包含SEQ ID NO:28和29任一所示的重链可变区序列，所述单克隆抗体包含SEQ ID NO:35所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:28所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:35所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:29所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:35所示的轻链可变区序列。
为了提高全人源抗体与人ADM的亲和力，本发明还设计和构建了全人源抗体的亲和力成熟文库。通过对全人源抗体亲和力成熟文库的筛选和单克隆的鉴定，本发明获得了不同的全人源抗ADM抗体，它们均展现出对ADM的KD值小于10-10M的亲和力。
因此，在本发明优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中：
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTX1Y，其中X1选自S、Q或H，
(ii)SX2YX3GX4，其中X2选自A或P，X3选自N、Q、S或T，X4选择N或K，
(iii)EGRX5GGSFX6I，其中X5选自S或W，X6选自D或N，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RAX7X8GIX9X10YLA，其中X7选自S或A，X8选自Q或E，X9选自S或G，X10选自S或E，
(ii)DX11SX12X13X14X15，其中X11选择A、V或T，X12选自N、I或D，X13 选自L或V，X14选自E或D，X15选自T或A，
(iii)QQYDX16LX17LX18，其中X16选自N或D，X17选自P或D，X18选自T或S。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTSY(如SEQ ID NO:43所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DASNLET(如SEQ ID NO:47所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTQY(如SEQ ID NO:51所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASEGISEYLA(如SEQ ID NO:52所示)，
(ii)DASNLET(如SEQ ID NO:47所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTQY(如SEQ ID NO:51所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RAAEGIGSYLA(如SEQ ID NO:53所示)，
(ii)DASNLET(如SEQ ID NO:47所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTSY(如SEQ ID NO:43所示)，
(ii)SPYSGN(如SEQ ID NO:54所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASEGISEYLA(如SEQ ID NO:52所示)，
(ii)DASNLET(如SEQ ID NO:47所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTHY(如SEQ ID NO:55所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DVSILDA(如SEQ ID NO:56所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTQY(如SEQ ID NO:51所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DVSILDA(如SEQ ID NO:56所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTQY(如SEQ ID NO:51所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DASNVDT(如SEQ ID NO:57所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTSY(如SEQ ID NO:43所示)，
(ii)SPYTGK(如SEQ ID NO:58所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DVSILDA(如SEQ ID NO:56所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTSY(如SEQ ID NO:43所示)，
(ii)SPYTGK(如SEQ ID NO:58所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DTSDLDT(如SEQ ID NO:59所示)，
(iii)QQYDNLPLT(如SEQ ID NO:48所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTHY(如SEQ ID NO:55所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DASNLET(如SEQ ID NO:47所示)，
(iii)QQYDDLDLT(如SEQ ID NO:60所示)。
在本发明进一步优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体 或其片段中，
(a)所述单克隆抗体的重链可变区包含以下CDR序列：
(i)GYTFTQY(如SEQ ID NO:51所示)，
(ii)SAYQGN(如SEQ ID NO:49所示)，
(iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
(b)所述单克隆抗体的轻链可变区包含以下CDR序列：
(i)RASQGISSYLA(如SEQ ID NO:46所示)，
(ii)DASNLET(如SEQ ID NO:47所示)，
(iii)QQYDDLPLS(如SEQ ID NO:61所示)。
此外，本发明还获得了亲和力提高后的抗体的重链可变区序列和轻链可变区序列。
由此，在本发明优选的实施方案中，在所述的抗肾上腺髓质素单克隆抗体或其片段中，所述的单克隆抗体包含SEQ ID NO:30至34任一所示的重链可变区序列，所述的单克隆抗体包含SEQ ID NO:35至42任一所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:30所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:35所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:31所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:36所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:31所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:37所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:32所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:36所示的轻链可变区序 列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:33所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:38所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:31所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:38所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:31所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:39所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:34所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:38所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:34所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:40所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:33所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:41所示的轻链可变区序列。
在本发明的某些实施方案中，所述人源化抗体包含SEQ ID NO:31所示的重链可变区序列，所述人源化抗体包含SEQ ID NO:42所示的轻链可变区序列。
在本发明的某些实施方案中，所述单克隆抗体还包含抗体轻链恒定区，所述轻链恒定区为人Kappa链恒定区。在本发明中，所述人Kappa链恒定区包含SEQ ID NO：20所示的氨基酸序列。
在本发明的某些实施方案中，所述单克隆抗体还包含抗体重链恒定区，所 述重链恒定区为人IgG1恒定区。在本发明中，所述人IgG1恒定区包含SEQ ID NO：21所示的氨基酸序列。
将所述的单克隆抗体的轻链可变区序列与人Kappa链恒定区组合成抗体轻链，将所述的单克隆抗体的重链可变区序列与人IgG1恒定区组合成抗体重链。
密码子优化后进行基因合成，克隆合成后基因片段到表达载体pcDNA3.4中，在CHO细胞表达、纯化，可以得到亲和力成熟的单克隆抗体。经亲和力测定，所述抗ADM抗体展现出对ADM的KD值小于10-10M的亲和力。
本发明再一方面提供了编码本发明所述的抗肾上腺髓质素单克隆抗体或其片段的核苷酸序列。
本发明再一方面提供了包含本发明所述的核苷酸序列的表达载体，其中优选为载体pcDNA3.4。
本发明再一方面提供了包含本发明所述的核苷酸序列或表达载体的宿主细胞，其中优选为CHO细胞。
本发明再一方面提供了一种包含本发明所述的抗肾上腺髓质素单克隆抗体或其片段的药物组合物、检测试剂或套装药盒。
本发明再一方面提供了本发明所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于维持内皮细胞完整性、增强屏障作用的药物中的应用。
本发明再一方面提供了本发明所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于控制血管舒张、降低血压的药物中的应用。
本发明再一方面提供了本发明所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于治疗或预防脓毒性休克或创伤性损伤，尤其是脓毒症晚期的药物中的应用。
本发明再一方面提供了本发明所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于降低患者的慢性或者急性病或急性病症的死亡风险的药物中的应用，所述慢性或者急性病或急性病症选自重度感染，如脑膜炎、全身炎症反应 综合征(SIRS)、败血症；其它疾病如糖尿病、癌症、急性和慢性血管疾病如心力衰竭、心肌梗死、卒中、动脉粥样硬化，水肿；休克如败血性休克和器官功能障碍如肾功能障碍、肝功能障碍、烧伤、手术、外伤、中毒。
本发明再一方面提供了本发明所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于稳定患者循环尤其是全身循环的药物中的应用，所述患者患有慢性或者急性病或急性病症，所述慢性或者急性病或急性病症选自重度感染，如脑膜炎、全身炎症反应综合征(SIRS)、败血症；其它疾病如糖尿病、癌症、急性和慢性血管疾病如心力衰竭、心肌梗死、卒中、动脉粥样硬化，水肿；休克如败血性休克和器官功能障碍如肾功能障碍、肝功能障碍、烧伤、手术、外伤、中毒。
在本发明优选的实施方案中，所述的抗肾上腺髓质素单克隆抗体或其片段与另一种药剂联合使用。
在本发明进一步优选的实施方案中，所述的药剂选自血管加压药，静脉注射液，TNF-α-抗体和抗生素。
技术效果
本发明通过杂交瘤技术筛选出抗ADM单克隆抗体，并通过抗体的人源化表达、全人源单链噬菌体抗体库淘选，获得了全人源抗ADM单克隆抗体。在此基础上，本发明通过筛选亲和力成熟文库，最终获得了具有较高的与人ADM的N端相结合的活性。与现有技术中存在的抗ADM非中和抗体相比，本发明的抗ADM非中和抗体展现出对ADM的KD值小于10-10M的亲和力。
动物试验表明，使用本发明的抗ADM非中和性抗体进行治疗，可以显著改善脓毒症症状，提高动物的存活率。
附图说明
图1是抗ADM抗体阻断hADM诱导CHOK1/CRLR/RAMP3细胞产生 cAMP示意图。
图2是抗ADM抗体对LPS诱导的小鼠脓毒症存活率结果图。
具体实施方式
为了更好的说明本方法的目的和优点，结合附图及具体实施例对本发明具体实施内容做进一步详细说明。
实施例1：杂交瘤技术筛选抗ADM单克隆
1.1小鼠免疫
分别将合成的人ADM的N端16个氨基酸多肽(简称YY-16，SEQ ID NO:1)、人ADM的N端21个氨基酸多肽(简称YY-21，SEQ ID NO:2)或鼠ADM的N端19个氨基酸多肽(简称YY-19，SEQ ID NO:3)与KLH(Sigma，H8283)的偶联物作为免疫原免疫小鼠。初次免疫时，将免疫原与弗氏完全佐剂以1:1的比例进行乳化，腹腔注射至6～8周龄雌性Balb/c小鼠、SJL小鼠或SD大鼠，100μg/只；之后每间隔2～3周进行加强免疫，每只动物50μg免疫原加弗氏不完全佐剂，免疫3～4次后采血测效价。最后一次免疫一周后取血，ELISA测定血清抗YY-21的滴度。血清效价高的鼠经腹腔注射50μg免疫原冲击免疫后，第三天取动物的脾脏细胞进行融合。
1.2脾细胞融合
鼠安乐死后，解剖、取脾、研磨并收集细胞，用5mL红细胞裂解液悬浮细胞，4℃放置5min，用DMEM+10％FBS终止反应。离心后用40mL DMEM重悬脾细胞，静置2～3min后转移上清到另外一个50mL离心管中。按SP2/0:脾细胞＝1:2的比例混合，离心，充分吸取上清后，混合细胞沉淀，用DMEM洗涤混合细胞两次，用电融合缓冲液重悬细胞，加入电融合槽中。待电融合程序结束后，将融合后的细胞先静置5min，再加入到DMEM+10％FBS+1×HAT筛选培养基中。将上述细胞悬液加入96孔细胞培养板内，置37℃、湿度 75％、5％CO2培养箱内培养7～9天。
1.3杂交瘤克隆的筛选
用PBS分别稀释YY-21或YY-19至1.0μg/mL，加至96孔板(Corning，9018)内，100μL/孔，置4℃包被过夜。第二天将ELISA板在自动洗板机上用洗涤缓冲液(PBS+0.05％吐温20)洗涤3次。每孔加入300μL封闭缓冲液(PBS+0.05％吐温20+1％BSA)，置室温封闭1h。然后在自动洗板机上用洗涤缓冲液洗涤3次，加杂交瘤上清至ELISA板各孔内，置室温孵育1h，然后依照上述方法洗板3次。用封闭缓冲液中1:5000稀释Goat Anti-mouse IgG Fc-HRP(Sigma，A0168)或Goat Anti-rat-IgG-HRP(Sigma，A5795)，每孔加入100μL，置室温孵育1h。然后依照上述方法洗板3次。加入100μL/孔TMB底物液室温孵育10min，然后每孔加入50μL 1.0M盐酸终止反应，通过酶标仪在OD450nm读板。挑取阳性细胞进行亚克隆及亚克隆筛选，直至得到稳定的可分泌结合YY-21和YY-19的单克隆抗体的杂交瘤细胞株。通过结合实验，筛选到杂交瘤克隆40E12，冻存细胞株，并采用无血清培养基进行50mL小规模生产，protein A柱纯化后做后续鉴定。
1.4杂交瘤克隆40E12的亲和力测定
采用Octet RED96e(Fortebio)测定候选抗体与生物素标记的人ADM(简称人ADM-C-biotin，人ADM序列如SEQ ID NO:4所示)和鼠ADM(简称鼠ADM-C-biotin，鼠ADM序列如SEQ ID NO:5所示)的亲和力。抗原及抗体均用1×PBST稀释，抗原使用浓度为2μg/ml，抗体使用工作浓度为100nM。
首先，将样品加入96孔板(Greiner bio-one，655209)，体系为200μL/well。然后设置软件参数，板温设定为30℃，收集标准动力学信号的频率为5.0HZ。接着，用1×PBST预湿链霉亲和素传感器(Fortébio，货号：18-5020)10min，然后上机检测。每个循环包含以下步骤：1)浸入缓冲液180s，使基线平稳；2)抗原固化10s，使抗原结合到传感器上，抗原结合量控制在0.5-1.0nm之间；3)传感器浸入缓冲液180s；4)抗原与抗体结合，结合时 间180s；5)抗原抗体的解离，时间10min。
采用Fortebio的Data Analysis 12.0软件，对抗原-抗体以1:1的结合方式，测定结合速率(Kon)和解离速率(Koff)，以此计算抗体的平衡解离常数(KD)结果如表1所示。
表1候选抗体40E12的平衡解离常数(KD)
1.5杂交瘤克隆40E12的测序
培养杂交瘤单克隆细胞株，离心收集5×106个杂交瘤细胞，Trizol法提取总RNA，逆转录反应后得到的cDNA，通过末端转移酶进行加G反应，后用VH引物(序列如SEQ ID NO:6所示)、VK引物(序列如SEQ ID NO:7所示)和polyC引物(序列如SEQ ID NO:8所示)扩增含可变区序列的DNA，做TA克隆，测序后得鼠源杂交瘤克隆40E12重链可变区(40E12VH)和40E12轻链可变区(40E12VK)序列分别如SEQ ID NO:9和SEQ ID NO:10所示。对轻重链可变区进行Chothia编号，采用Chothia定义的克隆40E12的HCDR1、HCDR2和HCDR3的氨基酸序列分别示于SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13，LCDR1、LCDR2和LCDR3的氨基酸序列分别示于SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16。
实施例2：抗ADM抗体的人源化和表达
2.1抗ADM抗体的人源化
通过序列比对，挑选同源性最高的人抗体胚系基因作为人源化设计框架。
重链可变区以人抗体胚系基因序列IGHV7-4-1*02和IGHJ6*01为框架，将40E12VH(又简称为2004hzVH0)进行CDR移植和回复突变获得人源化重链可变区序列2004hzVH9(氨基酸序列如SEQ ID NO:17)。轻链可变区以人抗体胚系基因序列IGKV2-30*02和IGKJ2*01为框架，将40E12VK(又简称为 2004hzVK0)进行CDR移植和回复突变的人源化轻链可变区序列2004hzVK7(氨基酸序列如SEQ ID NO:18)和2004hzVK9(氨基酸序列如SEQ ID NO:19)。
2.2抗ADM抗体的表达
将2004hzVK7、2004hzVK9序列与人Kappa链恒定区(CL，氨基酸序列SEQ ID NO:20)组合成抗体轻链，将2004hzVH9序列与人IgG1恒定区(CH，氨基酸序列SEQ ID NO:21)组合成抗体重链。2004hzVH0和2004hzVK0分别与人重链恒定区和人轻链恒定区组合后配对组成嵌合抗体2004hz00(序列如SEQ ID NO:22和23所示)；2004hzVH9和2004hzVK7分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体2004hz97(序列如SEQ ID NO:24和25所示)；2004hzVH9和2004hzVK9分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体2004hz99(序列如SEQ ID NO:26和27所示)。
密码子优化后进行基因合成，克隆合成后基因片段到表达载体pcDNA3.4(Life Technologies)。表达质粒扩增和质粒抽提(Qiagen,Plasmid Maxi Kit,Cat.No.12362)后双质粒共转Expi293F(ThermoFisher Scientific，A14527)或CHO-K1细胞(ECACC catalogue no.85051005)，根据供应商Expi293F或CHO-K1表达系统方法进行抗体瞬转表达，表达后通过Protein A亲和层析柱纯化，柠檬酸缓冲液(PH3.4)洗脱，使用NanoDrop仪器读取280nm吸光度值；透析收集抗体备用。
抗体表达结果如表2所示，表明利用上述方法能表达出抗ADM抗体。
表2候选抗体的表达量
实施例3：抗ADM抗体的亲和力测定
采用Octet RED96e(Fortebio)测定抗体2004hz00、2004hz97、2004hz99与生物素标记的人、小鼠ADM(人ADM货号：894757，小鼠ADM货号：894758，GLBiochem合成)的亲和力，抗原及抗体均用1×PBST(1×PBS：生工，B548117-0500；0.02％吐温20：sigma，P1379)稀释，抗体使用浓度为100nM，抗原使用浓度为2μg/mL。将候选抗体样品按200μL/孔加入96孔板(Greiner bio-one，655209)，设置软件参数，温度30℃、收集标准动力学信号的频率为5.0Hz；用1×PBST预湿SA传感器(Fortebio，货号：18-5020)10min，然后上机检测。
每个循环包含以下步骤：1)浸入缓冲液60s；2)检测抗原是否与传感器有非特异性结合；3)10mM pH1.7的甘氨酸溶液再生；4)浸入缓冲液60s；5)抗原固化在传感器上，时间为10s；6)传感器浸入缓冲液180s；7)抗原与抗体结合，时间180s；8)抗原抗体的解离，时间600s；9)传感器再生。
采用Fortebio的Data Analysis 12.0软件，对抗原-抗体以1:1的结合方式，测定结合速率(Kon)和解离速率(Koff)，以此计算抗体的平衡解离常数(KD)，结果分别如表3和表4所示。由表3和表4可知，人源化抗体2004hz97、2004hz99与人、小鼠ADM的亲和力与嵌合抗体2004hz00亲和力相当。
表3候选抗体与人ADM的亲和力
表4候选抗体与小鼠ADM的亲和力
实施例4：人源化抗体理化性质评估
抗体2004hz97和2004hz99的表达量及亲和力较为理想，作为候选分子继续进行理化成药性评估，具体如下。
4.1 SEC-HPLC纯度分析
将样品浓度调整至1mg/mL，离心取上清转至样品瓶，放入HPLC样品盘。设置色谱条件如下：色谱柱，TSK G3000SWxl；检测波长，280nm；柱温，25℃；样品室温度，5℃；流速，0.5mL/min。色谱柱采用流动相(200mM磷酸盐缓冲液，pH6.8)平衡后，进样分析，用色谱软件进行数据分析，峰面积归一化法计算各个峰的峰面积百分比，百分比越高说明抗体纯度越高。
4.2 HIC-HPLC分析
将样品浓度调整至1mg/mL，离心取上清待测。设置色谱条件如下：色谱柱，MAbPacTMHIC-10；检测波长，214nm；柱温，30℃；样品室温度，5℃；流速，0.8mL/min。用流动相A(50mM磷酸盐缓冲液/1M硫酸铵，pH7.0)和流动相B(50mM磷酸盐缓冲液，pH 7.0)进行梯度洗脱，记录主峰保留时间，出峰时间短则抗体亲水性强。
4.3熔解温度(Tm)值分析
按照Protein Thermal ShiftTM Starter Kit说明书，取供试品溶液13μL加入至PCR管内，加入5μL Protein Thermal shift TM Buffer，加入2μL10×染色液，使反应体积为20μL，混匀后，12000rpm离心5min以去除气泡。将检测样品置于PCR仪内，进行样品分析，记录样品的Tm值，Tm值越高表示抗体的热稳定性越好。
4.4等电聚焦(iCIEF)分析
取样品溶液加入到已经充分混匀的以下体系中：1％的甲基纤维素(MC)70μL，尿素5M 80μL，两性电解质Pharmalyte pH 3-10 8μL，pI marker 5.5和9.5各2μL。补加适当体积超纯水至200μL，混匀。离心取上清进样分析。分 析结束后，将结果文件导入ChromPerfect软件进行图谱积分处理并计算各峰的等电点以及各峰百分比，分析候选抗体的电荷异构体分布情况。
4.5 nrCE-SDS
用超纯水将样品稀释到4mg/mL，取25μL，加入SDS样品缓冲液75μL，0.25mol.L-1碘乙酰胺(IAM)5μL，混匀后，70℃加热10min，取90μL上清放入样品瓶中上机分析。采用Beckman PA800Plus毛细管电泳系统，无涂层毛细管(总长度31cm，有效长度21cm)检测；检测条件：分离电压为15KV，毛细管温度25℃，样品室温度15℃，检测波长220nm。计算主峰校正峰面积百分比。
由表5可知，人源化抗体2004hz97、2004hz99经过一步protein A纯化，已具有良好的理化特性。
表5候选抗体理化性质分析结果
实施例5:全人源单链噬菌体抗体库淘选
取150μL Streptavidin Magnetic Beads(Thermo fisher，货号：88817)与2mL全人源单链噬菌体抗体库预结合，室温孵育90min，去除非特异性结合。将去除背景后的文库噬菌体加入10μg人ADM(货号NT-H-2，金斯瑞合成)，150μL Streptavidin Magnetic Beads，室温孵育15min，PBST(PBS中含有0.05％Tween-20)洗14遍，洗去不结合的噬菌体。用450μL 100mM盐酸洗脱抗原特异性结合的噬菌体，加入50μL pH11的1M Tris-HCl中和并感染处于对数生长期的大肠杆菌SS320，产生并纯化噬菌体用于下一轮的筛选。筛选方法与第一轮相同，仅将抗原用量减为4μg。取两轮筛选后富集的噬菌体用酶联免疫(ELISA)鉴定富集情况，结果表明，经过两轮淘选后噬菌体富集明显。
取10μL两轮淘选后洗脱的噬菌体梯度稀释10000倍，加入90μL对数生 长期的大肠杆菌SS320，静置侵染30min后涂布抗性平板，37℃过夜培养。次日从抗性平板上挑取单克隆菌斑放入已加入氨苄青霉素/IPTG/2YT的96深孔板中，37℃过夜培养。次日4000g离心10min取上清，通过酶联免疫(ELISA)鉴定单克隆结合能力筛选出单克隆1F12(可变区氨基酸序列如SEQ ID NO:28和35所示)。
实施例6：抗ADM抗体对ADM生物活性的影响
在人重组肾上腺髓质素受体cAMP功能测定(肾上腺髓质素生物测定)中检测所选的抗ADM抗体对ADM生物活性的影响。
用Stimulation Buffer 1(Cisbio，64SB1FDD)将抗ADM抗体稀释到1600μg/mL，工作浓度为400μg/mL，随后用Stimulation Buffer 1进行3倍梯度稀释(8μL+16μL Stimulation Buffer 1)，其中拮抗剂人ADM(22-52)(Alfa Aesar，货号：159899-65-7)稀释到12000μg/mL，工作浓度为3000μg/mL，随后实验板中相应孔加入2.5μL抗ADM抗体。将人ADM JMB2004 YY-52蛋白(吉尔生化，货号：196191)用Stimulation Buffer 1稀释到0.6μg/mL，相应孔加入2.5μL，即人ADM JMB2004 YY-52蛋白工作浓度为0.15μg/mL，随后实验板于室温孵育60min。用TrypLE Express(gibco，货号：12604-021)消化分离表达人重组肾上腺髓质素受体的CHO-K1细胞(以下简称CHO-K1/CRLR/RAMP3，其中CRLR的基因登录号为U17473,RAMP3的基因登录号为AJ001016)，通过离心收集并重悬于Stimulation Buffer 1中，调整细胞密度至4×106/mL，每孔加入5μL CHO-K1/CRLR/RAMP3细胞(2×104细胞/孔)。随后实验板于37℃细胞培养箱孵育90min后，用HTRF试剂盒(Cisbio，62AM4PEB)检测cAMP的含量，即实验孔加入5μL cAMP-d2 reagent工作液，然后加入5μL cAMP Eu-Cryptate antibody working solution，室温孵育1h后，酶标仪检测HTRF值。
实验结果如图1所示，从图1可以看出，抗体1F12无阻断活性。
实施例7：全人源抗体1F12的亲和力成熟文库设计与构建
对筛选获得的全人源抗体1F12进行亲和力成熟，提高其与人ADM的亲和力。同时，由于1F12的重链抗原结合决定簇2(CDR_H2)含有NG的翻译后修饰(PTM)位点，定点突变成QG去除脱酰胺异构作用，突变的抗体命名为1F12PTMΔ(可变区氨基酸序列如SEQ ID NO:29和35所示)。以突变体1F12PTMΔ为亲本，按Chothia规则进行编码，按Chothia定义CDR区，对其HCDR-3、LCDR-3位点的氨基酸进行随机突变，构建突变文库。设计NNK突变引物进行聚合酶链式反应(PCR)扩增HCDR-3、LCDR-3突变文库基因片段，扩增的VH、VL基因片段回收后与酵母展示质粒共同电转入酿酒酵母菌株EBY100(购自ATCC)，通过酿酒酵母的同源重组使VH、VL基因插入至酵母展示质粒中，进而实现在酵母细胞壁表面展示抗体的Fab突变文库，文库命名为JYYDL196-197。电转后文库JYYDL196-197在250mL的SD-Trp-Leu液体培养基(Clontech，货号：630316)，30℃培养过夜；各取1.0×109菌量，重悬于200mL YPGP诱导培养基(2％半乳糖，2％蛋白胨，1％酵母提取物，0.54％Na2HPO4，0.86％NaH2PO4·H2O)，20℃培养24h，置于4℃待用。同时将亲本1F12PTMΔ的序列展示于酵母表面，作为亲本对照使用。
实施例8：全人源抗体1F12亲和力成熟文库筛选与单克隆鉴定
JYYDL196-197文库诱导后菌液，测定菌液的OD600，按1OD为1.0×107细胞数计算，取1.0×109细胞，用磁珠分选系统进行第一轮富集：用50mL1×PBSA(1×PBS+1％BSA)洗涤一次，离心弃上清；与5mL含100nM生物素标记的人ADM(简称hADM-Biotin，货号NT-H-2,金斯瑞合成)的1×PBSA，室温孵育30min；洗涤后加入抗生物素的磁珠(miltenyi，货号：130-090-485)混匀孵育10min，过磁力柱(Quadro MACS Starting Kit)收集阳性细胞。阳性细胞经过再次培养、诱导后，取3.0×107细胞进行第二轮流式分 选：用1mL 1×PBSA，离心弃上清；与1mL含10nM hADM-Biotin及鼠抗V5抗体(Invitrogen，货号2156578，1:1000稀释)的1×PBSA冰上孵育30min；离心弃上清，加入1mL 1×PBSA洗涤一次；加入500μL含荧光抗体的1×PBSA(SA-PE厂家eBioscience，货号：12-4317-8，按1:200稀释；羊抗鼠-647厂家Invitrogen，货号：A21235，按1:400稀释)，避光冰上孵育20min；洗涤后加入2mL 1×PBSA重悬细胞，通过流式分选仪器收集647荧光信号与PE荧光信号均强的细胞群。第二轮流式分选后细胞经过再次培养、诱导后，同第二轮方法，取3.0×107细胞在3nM hADM-Biotin孵育后进行第三轮流式分选，分选后取部分细胞涂布于SD-Trp-Leu固体培养基(Clontech，货号：630317)平板，30℃静置培养3天。
JYYDL196-197第三轮筛选产物，各挑取92个单克隆进行测序分析，最终获得独一序列的酵母单克隆菌落进行流式染色分析，各取1×106个细胞进行染色评估，根据各克隆的染色结果，综合各克隆序列的相似性，最终挑取全人源抗体Ab2004.Am01(可变区氨基酸序列如SEQ ID NO:30和35所示)进行表达。
实施例9：Ab2004.Am01亲和力成熟文库设计与构建
对Ab2004.Am01继续进行亲和力成熟，提高其与人、小鼠ADM的亲和力。参考实施例7，选择Ab2004.Am01的HCDR-1、HCDR-2、LCDRL-1、LCDR-2、LCDR-3的氨基酸进行突变文库构建，文库编号JYYDL208-212。文库JYYDL208-212培养、诱导后待用。同时将Ab2004.Am01的Fab序列展示于酵母表面，作为本轮亲和成熟的亲本对照使用。
实施例10：Ab2004.Am01亲和力成熟文库筛选
JYYDL208-212文库诱导后菌液，取1.0×109细胞用磁珠分选系统进行第一轮富集，磁珠筛选后阳性细胞经过再次培养、诱导，取3.0×107细胞进行第二 轮流式分选，通过流式分选仪器收集647荧光信号与PE荧光信号均强的细胞群，细胞群培养、诱导后进行第三轮流式筛选。JYYDL208-209的第二轮筛选产物，取3.0×107细胞在10nM mADM-Biotin条件下进行第三轮流式分选；JYYDL210-212取3.0×107细胞在100nM mADM-Biotin条件下进行第三轮流式分选。第二轮、第三轮分选后细胞涂布于SD-Trp-Leu固体培养基平板，30℃静置培养3天。
实施例11：轻、重链突变组合文库构建与筛选
JYYDL208-209，JYYDL211第二轮、第三轮筛选产物，各挑取若干单克隆构建轻、重链突变组合文库JYYDL227。
JYYDL227取2.0×107细胞，分别用3nM hADM-Biotin和1.2nM mADM-Biotin进行流式第一轮分选；分选后取细胞涂布于SD-Trp-Leu固体培养基，30℃静置培养3天。
JYYDL227第一轮筛选产物，各挑取46个单克隆进行测序，最终获得独一序列的酵母单克隆菌落进行流式染色EC50鉴定。各取1×105个细胞进行染色评估：1、评估各克隆在不同抗原浓度下与人ADM的结合水平，计算出各克隆与人ADM的EC50值，值越小说明亲和力越强；2、评估各克隆在不同抗原浓度下与小鼠ADM的结合水平，同理可得到各克隆与小鼠ADM的亲和力强弱。
根据各克隆的染色结果(如表6所示)，综合各克隆序列的相似性，最终挑取了表达抗体Ab2004.Am31(可变区氨基酸序列如SEQ ID NO:31和36所示)、Ab2004.Am32(可变区氨基酸序列如SEQ ID NO:31和37所示)、Ab2004.Am33(可变区氨基酸序列如SEQ ID NO:32和36所示)、Ab2004.Am34(可变区氨基酸序列如SEQ ID NO:33和38所示)、Ab2004.Am35(可变区氨基酸序列如SEQ ID NO:31和38所示)、Ab2004.Am36(可变区氨基酸序列如SEQ ID NO:31和39所示)、Ab2004.Am37(可变区氨基酸序列如SEQ ID NO:34和38所示)、 Ab2004.Am38(可变区氨基酸序列如SEQ ID NO:34和40所示)、Ab2004.Am39(可变区氨基酸序列如SEQ ID NO:33和41所示)和Ab2004.Am40(可变区氨基酸序列如SEQ ID NO:31和42所示)的单克隆。候选抗体的Chothia编号的CDR区氨基酸序列见表7，候选抗体的轻重链可变区氨基酸序列见表8。
表6酵母单克隆菌落流式染色结果
表7候选抗体的Chothia编号的CDR序列

表8候选抗体的可变区序列
实施例12：候选抗体的表达
同实施例2.2，将各克隆的VH与VK序列分别与人Kappa链恒定区(CL，氨基酸序列SEQ ID NO:20)和人IgG1恒定区(CH，氨基酸序列SEQ ID NO:21)组合成抗体轻链和重链，装入表达载体pcDNA3.4(Life  Technologies)，委托南京金斯瑞生物科技有限公司进行瞬转CHO细胞表达、纯化，最终得到亲和力成熟候选抗体见表9。
表9亲和力成熟候选抗体表达、纯化数据
实施例13：候选抗体亲和力测定
同实施例3，继续测定亲和力成熟抗体Ab2004.Am31-Ab2004.Am40及亲本抗体Ab2004.Am01分别与人、小鼠ADM的亲和力，结果如表10。由表10可知，Ab2004.Am31、Ab2004.Am34、Ab2004.Am39与人ADM的亲和力相对于亲本抗体Ab2004.Am01提高最大，且与小鼠ADM的亲和力与阳性对照抗体Enibarcimab相当。
表10候选抗体与人、小鼠ADM的亲和力测定

实施例14：候选抗体理化性质评估
候选抗体Ab2004.Am31、Ab2004.Am34、Ab2004.Am39的表达量及亲和力较为理想，继续进行理化成药性评估，结果汇总如表11。由表11可知，Ab2004.Am31、Ab2004.Am34、Ab2004.Am39在纯度、热稳定性、亲水性、电荷异构体等方面均符合成药性标准，方法参考实施例4。
表11亲和力成熟抗体理化性质分析结果
实施例15：ADM抗体在LPS诱导的C57BL/6J小鼠脓毒症模型中的药效学研究
C57BL/6J小鼠购自北京维通利华实验动物技术有限公司，10-11周龄，雄性，共50只。按照体重将动物随机分成以下五组(n＝10)：G1组为模型组给予同型对照RSV-IgG1(2mg/kg，IV，Single)，G2组为阳性对照组给予Enibarcimab(2mg/kg，IV，Single)，G3、G4、G5组则分别给予本发明抗体2004hz97(2mg/kg，IV，Single)、2004hz99(2mg/kg，IV，Single)和Ab2004.Am34(2mg/kg，IV，Single)。LPS诱导脓毒症前5min单次IV给予相应抗体治疗，5min后单次IP给予20mg/kg的LPS(E.coli 055:B5；Sigma)诱导小鼠脓毒症，造模后每天2次、连续7天观察动物死亡情况。数据采用GraphPad Prism 8软件作图，并用Log-rank(Mantel-Cox)test方法对数据进行统计学分析。
LPS处理7天后各组动物存活率分别为：RSV-IgG1(40％)、Enibarcimab (50％，P＝0.6713vs RSV-IgG1)、2004hz97(90％，P<0.05vs RSV-IgG1)、2004hz99(80％，P＝0.1001vs RSV-IgG1)、Am34(80％，P<0.05vs RSV-IgG1)结果显示使用本发明抗体治疗后可显著提高动物存活率，改善脓毒症症状(参见表12，附图2)。
表12候选抗体对LPS诱导的小鼠脓毒症存活率
以上所述为本发明的较佳实施例而已，本发明不应该局限于该实施例和附图所公开的内容。凡是不脱离本发明所公开的精神下完成的等效或修改，都落入本发明保护的范围。
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Claims (35)

  1. 一种抗肾上腺髓质素(抗ADM)单克隆抗体或其片段，其中所述单克隆抗体或片段特异性结合人肾上腺髓质素(ADM)的N端第1-21个氨基酸序列，所述人ADM的N端第1-21位氨基酸序列如SEQ ID NO：2所示，并且所述单克隆抗体或片段展现出对ADM的KD值小于10-10M的亲和力。
  2. 根据权利要求1所述的抗肾上腺髓质素单克隆抗体或其片段，其中所述片段包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
  3. 根据权利要求1或2所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYAFTTF(如SEQ ID NO:11所示)，
    (ii)NTYSRV(如SEQ ID NO:12所示)
    (iii)GYGGEGGLGF(如SEQ ID NO:13所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RSSQSIIDSDGNTYLE(如SEQ ID NO:14所示)，
    (ii)KVSNRFS(如SEQ ID NO:15所示)，
    (iii)FQGSHFPYT(如SEQ ID NO:16所示)。
  4. 根据权利要求3所述的抗肾上腺髓质素单克隆抗体或其片段，其中所述单克隆抗体包含SEQ ID NO:9和17任一所示的重链可变区序列，所述单克隆抗体包含SEQ ID NO:10、18和19任一所示的轻链可变区序列，其中所述单克隆抗体优选包含SEQ ID NO:9所示的重链可变区序列和SEQ ID NO:10所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:17所示的重链可变区序列和SEQ ID NO:18所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:17所示的重链可变区序列和SEQ ID NO:19所示的轻链可变区序列。
  5. 根据权利要求4所述的抗肾上腺髓质素单克隆抗体或其片段，其中所述单克隆抗体包含SEQ ID NO:23所示的重链序列和SEQ ID NO:22所示的轻链序列。
  6. 根据权利要求4所述的抗肾上腺髓质素单克隆抗体或其片段，其中所述单克隆抗体包含SEQ ID NO:25所示的重链序列和SEQ ID NO:24所示的轻链序列。
  7. 根据权利要求4所述的抗肾上腺髓质素单克隆抗体或其片段，其中所述单克隆抗体包含SEQ ID NO:27所示的重链序列和SEQ ID NO:26所示的轻链序列。
  8. 根据权利要求1或2所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTX1Y，其中X1选自S、Q或H，
    (ii)SX2YX3GX4，其中X2选自A或P，X3选自N、Q、S或T，X4选择N或K
    (iii)EGRX5GGSFX6I，其中X5选自S或W，X6选自D或N，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RAX7X8GIX9X10YLA，其中X7选自S或A，X8选自Q或E，X9选自S或G，X10选自S或E，
    (ii)DX11SX12X13X14X15，其中X11选择A、V或T，X12选自N、I或D，X13选自L或V，X14选自E或D，X15选自T或A，
    (iii)QQYDX16LX17LX18，其中X16选自N或D，X17选自P或D，X18选自T或S。
  9. 根据权利要求8所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含HCDR1、HCDR2和HCDR3序列，其中所述HCDR1包含SEQ ID NO:43、51和55任一所示的氨基酸序列，所述HCDR2包含SEQ ID NO:44、49、54和58任一所示的氨基酸序列，所述HCDR3包含SEQ ID NO:45和50任一所示的氨基酸序列，以及
    (b)所述单克隆抗体的轻链可变区包含LCDR1、LCDR2和LCDR3序列，其中所述LCDR1包含SEQ ID NO:46、52和53任一所示的氨基酸序列，所述LCDR2包含SEQ ID NO:47、56、57和59任一所示的氨基酸序列，所述LCDR3包含SEQ ID NO:48、60和61任一所示的氨基酸序列。
  10. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTSY(如SEQ ID NO:43所示)，
    (ii)SAYNGN(如SEQ ID NO:44所示)，
    (iii)EGRSGGSFDI(如SEQ ID NO:45所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DASNLET(如SEQ ID NO:47所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  11. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTSY(如SEQ ID NO:43所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRSGGSFDI(如SEQ ID NO:45所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DASNLET(如SEQ ID NO:47所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  12. 根据权利要求8至11中任一项所述的抗肾上腺髓质素单克隆抗体或其片段，其中所述单克隆抗体包含SEQ ID NO:28或29所示的重链可变区序列，所述单克隆抗体包含SEQ ID NO:35所示的轻链可变区序列，其中所述单克隆抗体优选包含SEQ ID NO:28所示的重链可变区序列和SEQ ID NO:35所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:29所示的重链可变区序列和SEQ ID NO:35所示的轻链可变区序列。
  13. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTSY(如SEQ ID NO:43所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DASNLET(如SEQ ID NO:47所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  14. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTQY(如SEQ ID NO:51所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASEGISEYLA(如SEQ ID NO:52所示)，
    (ii)DASNLET(如SEQ ID NO:47所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  15. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTQY(如SEQ ID NO:51所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RAAEGIGSYLA(如SEQ ID NO:53所示)，
    (ii)DASNLET(如SEQ ID NO:47所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  16. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTSY(如SEQ ID NO:43所示)，
    (ii)SPYSGN(如SEQ ID NO:54所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASEGISEYLA(如SEQ ID NO:52所示)，
    (ii)DASNLET(如SEQ ID NO:47所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  17. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTHY(如SEQ ID NO:55所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DVSILDA(如SEQ ID NO:56所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  18. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTQY(如SEQ ID NO:51所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DVSILDA(如SEQ ID NO:56所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  19. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTQY(如SEQ ID NO:51所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DASNVDT(如SEQ ID NO:57所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  20. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTSY(如SEQ ID NO:43所示)，
    (ii)SPYTGK(如SEQ ID NO:58所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DVSILDA(如SEQ ID NO:56所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  21. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTSY(如SEQ ID NO:43所示)，
    (ii)SPYTGK(如SEQ ID NO:58所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DTSDLDT(如SEQ ID NO:59所示)，
    (iii)QQYDNLPLT(如SEQ ID NO:48所示)。
  22. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTHY(如SEQ ID NO:55所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DASNLET(如SEQ ID NO:47所示)，
    (iii)QQYDDLDLT(如SEQ ID NO:60所示)。
  23. 根据权利要求9所述的抗肾上腺髓质素单克隆抗体或其片段，其中：
    (a)所述单克隆抗体的重链可变区包含以下CDR序列：
    (i)GYTFTQY(如SEQ ID NO:51所示)，
    (ii)SAYQGN(如SEQ ID NO:49所示)，
    (iii)EGRWGGSFNI(如SEQ ID NO:50所示)，以及
    (b)所述单克隆抗体的轻链可变区包含以下CDR序列：
    (i)RASQGISSYLA(如SEQ ID NO:46所示)，
    (ii)DASNLET(如SEQ ID NO:47所示)，
    (iii)QQYDDLPLS(如SEQ ID NO:61所示)。
  24. 根据权利要求8至9、13至23中任一项所述的抗肾上腺髓质素单克隆抗体或其片段，其中所述单克隆抗体包含SEQ ID NO:30至34任一所示的重链可变区序列，所述单克隆抗体包含SEQ ID NO:35至42任一所示的轻链可变区序列，其中所述单克隆抗体优选包含SEQ ID NO:30所示的重链可变区序列和SEQ ID NO:35所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:31所示的重链可变区序列和SEQ ID NO:36所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:31所示的重链可变区序列和SEQ ID NO:37所示的轻链可变区序列，所述单克隆抗体优选包含SEQ ID NO:32所示的重链可变区序列和SEQ ID NO:36所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:33所示的重链可变区序列和SEQ ID NO:38所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:31所示的重链可变区序列和SEQ ID NO:38所示的轻链可变区序列，所述单克隆抗体优选包含SEQ ID NO:31所示 的重链可变区序列和SEQ ID NO:39所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:34所示的重链可变区序列和SEQ ID NO:38所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:34所示的重链可变区序列和SEQ ID NO:40所示的轻链可变区序列，所述单克隆抗体优选包含SEQ ID NO:33所示的重链可变区序列和SEQ ID NO:41所示的轻链可变区序列，所述单克隆单体优选包含SEQ ID NO:31所示的重链可变区序列和SEQ ID NO:42所示的轻链可变区序列。
  25. 编码权利要求1至24中任一项所述的抗肾上腺髓质素单克隆抗体或其片段的核苷酸序列。
  26. 包含权利要求25所述的核苷酸序列的表达载体，其优选为pcDNA3.4。
  27. 包含权利要求25所述的核苷酸序列或权利要求26所述的表达载体的宿主细胞，其优选为CHO细胞。
  28. 包含权利要求1至24中任一项所述的抗肾上腺髓质素单克隆抗体或其片段的药物组合物、检测试剂或套装药盒。
  29. 权利要求1至24中任一项所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于维持内皮细胞完整性、增强屏障作用的药物中的应用。
  30. 权利要求1至24中任一项所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于控制血管舒张、降低血压的药物中的应用。
  31. 权利要求1至24中任一项所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于治疗或预防脓毒性休克或创伤性损伤，尤其是脓毒症晚期的药物中的应用。
  32. 权利要求1至24中任一项所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于降低患者的慢性或者急性病或急性病症的死亡风险的药物中的应用，所述慢性或者急性病或急性病症选自重度感染，如脑膜炎、全身炎症反应综合征(SIRS)、败血症；其它疾病如糖尿病、癌症、急性和慢性血管疾病如心力衰竭、心肌梗死、卒中、动脉粥样硬化，水肿；休克如败血性休克和器官功能障碍如肾功能障碍、肝功能障碍、烧伤、手术、外伤、中毒。
  33. 权利要求1至24中任一项所述的抗肾上腺髓质素单克隆抗体或其片段在制备用于稳定患者循环尤其是全身循环的药物中的应用，所述患者患有慢性或者急性病或急性病症，所述慢性或者急性病或急性病症选自重度感染，如脑膜炎、全身炎症反应综合征(SIRS)、败血症；其它疾病如糖尿病、癌症、急性和慢性血管疾病如心力衰竭、心肌梗死、卒中、动脉粥样硬化，水肿；休克如败血性休克和器官功能障碍如肾功能障碍、肝功能障碍、烧伤、手术、外伤、中毒。
  34. 权利要求29至33所述的应用，其中所述抗肾上腺髓质素单克隆抗体或其片段与另一种药剂联合使用。
  35. 权利要求34所述的应用，其中所述药剂选自血管加压药，静脉注射液，TNF-α-抗体和抗生素。
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